Rickettsia parkeri in Amblyomma triste from Uruguay.

Our goal was to detect whether spotted fever group Rickettsia are found in the suspected vector of rickettsioses, Amblyomma triste, in Uruguay. Rickettsia parkeri was detected in A. triste, which suggests that this species could be considered a pathogenic agent responsible for human rickettsioses in Uruguay.

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In South America, cases of rickettsioses produced by the genus Rickettsia have been described in several countries in the last 20 years. The first three native cases of rickettsioses in Uruguay were reported in 1990. Patients had an initial small necrotic lesion (eschar) on the tick-bite point of attachment, fever and regional lymphadenopathies, an erythematous maculopapular rash, or any combination of these symptoms. Ticks involved in these cases were classified as Amblyomma triste (1), formerly thought to be A. maculatum (2).

A. triste is a neotropical tick species with a variety of hosts (3,4). It is the main tick species feeding on humans in Uruguay, and it is the primary candidate vector for transmitting rickettsioses in this country (5). According to the literature (2), Rickettsia conorii has been the causative agent of rickettsial diseases in Uruguay, but the evidence has been only serologic (by antirickettsial microimmunofluorescence testing) in all patients with suspected rickettsioses (6,7). Neither rickettsial isolation nor polymerase chain reaction (PCR) amplification from human blood samples from patients from Uruguay have been performed. However, as has been suggested (8), other tick-transmitted rickettsiae could be present in Uruguay.

The Study

The aim of this study was to identify the spotted fever group (SFG) rickettsial species present in the suspected vector of SFG rickettsioses in Uruguay (A. triste). From 1999 to 2004, in Uruguay, ticks were collected from humans (with and without rickettsial syndrome), other mammals, and vegetation and preserved in ethanol 70% at room temperature. Species, sex, and stage of development were determined by members of the Facultad de Veterinaria, Universidad de la Republica (Uruguay). Classified adult ticks (N = 91) were sent to the Hospital de La Rioja (Spain) for analysis with molecular biologic techniques. Thirty-six ticks recovered from 14 humans were attached but nonengorged. Only one tick removed from a human, the one corresponding to human 3, was attached and engorged. A total of 16 A. triste were captured walking on three different humans (nonattached). The remaining ticks were attached to two goats (n = 3), a rodent of the species Scapteromys tumidus (n = 4), and three dogs (n = 30; 19 of them were engorged). One tick was recovered from vegetation. Details are shown in the Table.

DNA from the ticks was extracted by using the Tissue DNA Spin Kit (Genomed, Granada, Spain) according to the manufacturer's instructions. PCR testing for ompA, gltA, and 16S rRNA genes was performed as previously described (9-11). Two negative controls (one of them with template DNA but without primers and the other with primers and containing water instead of template DNA) as well as a positive control (R. conorii Malish #7 grown in Vero cells) were included in all PCR assays. Restriction analysis of ompA amplicons was also carried out under conditions reported by Roux et al. (12). Each PCR-amplifled fragment of ompA gene was sequenced twice for all positive samples (Universidad de Alcala de Henares, Spain) to confirm the identification of rickettsiae. Data were aligned with homologous sequences of reference strains of the SFG rickettsiae retrieved from the GenBank database.

Six ticks (three females and three males) collected on three humans and three dogs yielded positive PCR products of the expected sizes for ompA, gltA, and 16S rRNA, respectively (Table). One of these ticks infected with SFG Rickettsia (the only one that was engorged) was removed from a woman (human 3) diagnosed with rickettsial syndrome in the Instituto de Higiene, Facultad de Medicina, Universidad de la Republica (Uruguay). This patient showed a small initial maculopapulous lesion on her scalp at the tick-bite point, followed by regional lymphadenopathies and fever. Diagnosis was made on the basis of the clinical picture and indirect immunoglobulin (Ig) G immunofluorescent technique with R. conorii antigen (Biomerieux Laboratories, Marcy l'Etiole, France). Serum specimens were collected during the acute phase (day 0) and convalescent phase (1 month later). The patient showed seroconversion for R. conorii with IgG, and she had a benign disease course after treatment with oral tetracyclines. No clinical signs of infection were confirmed for the remaining two humans bitten by ticks infected with SFG Rickettsia (humans 6 and 7), but ticks were removed immediately after attachment in these cases. For all six positive samples, sequence analysis for ompA amplicons showed 100% similarity with the homologous sequence of R. parkeri (GenBank accession no. U43802). Profiles obtained with RsaI for ompA PCR fragments were also in accordance with these data.

Conclusions

SFG Rickettsia isolated from artbropods and initially classified as nonpathogenic to humans are increasingly recognized as causing emerging rickettsial diseases (13). In the last 10 years, different Rickettsia species and subspecies, such as R. aeschlimannii (14), R. sibirica strain mongolotimonae (15), and R. slovaca (16), among others, have been implicated as human pathogens. Very recently, a new tickborne Rickettsia, R. parkeri, has been identified as a cause of human disease in the southern United States (17). According to Paddock et al., R. parkeri rickettsioses may also occur in other regions of the Western Hemisphere, e.g., in Uruguay.

We report R. parkeri infection in A. triste ticks collected in Uruguay. Several cases of rickettsioses have been described in this country but, to date, no Rickettsia has been isolated, cultivated, and characterized as the causative agent. A few years ago, R. conorii was presumptively considered the etiologic agent, but diagnosis was established with serologic assays (indirect microimmunofluorescence testing) as reference technique (6). Cross-reactions are noted within SFG Rickettsia antigens, and available serologic tests cannot be used to implicate a specific pathogen. In Uruguay, A. triste frequently bites humans, and rickettsioses frequently develop in them (5). Our finding of R. parkeri infection in one A. triste tick collected from a patient with rickettsiosis suggests that R. parkeri could be a pathogenic SFG Rickettsia involved in rickettsial diseases in Uruguay. Traditionally, this agent was reported as nonpathogenic to humans, but the first report of a human infection with R. parkeri was recently published (17). It has also recently shown to be mildly pathogenic to guinea pigs (18). In our study, R. parkeri was the only detected SFG Rickettsia in A. triste ticks from Uruguay. Our data suggest that A. triste is a host of SFG Rickettsia in Uruguay, and R. parkeri could be the causative agent of human cases of rickettsioses in Uruguay.

Table. Amblyomma triste ticks collected from different
origins in Uruguay (a)

                No. of ticks
                  (N = 91)

                                              Date of
Host          Males   Females    Location    isolation

Human 1         0        1      Maldonado    Nov 1999
Human 2         2        0      Canelones    Oct 2000
Human 3 (b)     1        0      Montevideo   Oct 2000
Human 4         2        0      Maldonado    Dec 2000
Human 5         1        0       San Jose    Oct 2001
Human 6         0        1      Canelones    Sep 2002
Human 7         1        0      Montevideo   Dec 2002
Human 8         1        2      Montevideo   Oct 2002
Human 9         2        7      Montevideo   Oct 2002
Human 10        1        1      Canelones    Aug 2003
Human 11        1        1      Canelones    Aug 2003
Human 12        2        1      Montevideo   Oct 2003
Human 13        0        1      Montevideo   Sep 2003
Human 14        3        4      Canelones    Sep 2003
Human 15        4        8      Montevideo   Oct 2003
Human 16        2        0      Canelones    Nov 2003
Human 17        1        0      Canelones    Nov 2003
Human 18        0        2      Montevideo   Jan 2004
Goat 1          1        0      Maldonado    Nov 1999
Goat 2          0        2      Canelones    Oct 2000
Rodent          2        2      Montevideo   Oct 2000
Dog 1           3       21      Maldonado    Dec 2000
Dog 2           0        1       San Jose    Oct 2001
Dog 3           1        4      Canelones    Sep 2002
Vegetation      0        1      Montevideo   Dec 2002

                 PCR amplification     SFG Rickettsia
                                       species found
Host          ompA   gltA   16S rRNA      in ticks

Human 1        -      -        -
Human 2        -      -        -
Human 3 (b)    +      +        +         R. parkeri
Human 4        -      -        -
Human 5        -      -        -
Human 6        +      +        +         R. parkeri
Human 7        +      +        +         R. parkeri
Human 8        -      -        -
Human 9        -      -        -
Human 10       -      -        -
Human 11       -      -        -
Human 12       -      -        -
Human 13       -      -        -
Human 14       -      -        -
Human 15       -      -        -
Human 16       -      -        -
Human 17       -      -        -
Human 18       -      -        -
Goat 1         -      -        -
Goat 2         -      -        -
Rodent         -      -        -
Dog 1         +(3)   +(3)     +(3)       R. parkeri
Dog 2          -      -        -
Dog 3          -      -        -
Vegetation     -      -        -

(a) PCR, polymera se chain reaction; SFG, spotted fever group.

(b) Human 3 had rickettsioses.

Acknowledgments

We thank Fatima Bacellar for supplying SFG Rickettsia-positive controls and Elena Zanetta for providing clinical data.

This study was partly supported by grants from the Fondo de lnvestigacion Sanitaria (FIS PI021810 y FIS G03/057) from the Ministerio de Sanidad y Consumo, Spain.

References

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(4.) Venzal JM. Gonzalez EM, Capellino D, Estrada Pena A, Guglielmone AA First record of Amblyomma triste Koch. 1844 (Acari: Ixodidae) and new records of Ornithodoros mimon Kohls, Clifford & Jones, 1969 (Acari: Argasidue) from neotropical bats. Syst Appl Acarol. 2003;8:93-6.

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(12.) Roux V, Fournier P, Raoult D. Differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of PCR amplified DNA of the gene encoding the protein rOmpA. J Clin Microbiol. 1996;34:2058-65.

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Dr. Venzal is a researcher in the Veterinary Parasitology Department at Universidad de La Republica, Montevideo, Uruguay. His research interests include ticks (Ixodida) and the epidemiology of tickborne diseases.

Address for correspondence: Jose M. Venzal, Departamento de Parasitologia Veterinaria, Facultad de Veterinaria, Universidad de la Republica, Av. Alberto Lasplaces, 1550, 11600- Montevideo, Uruguay; fax: [phone]; email: [e-mail]

Jose M. Venzal, * Aranzazu Portillo, ([dagger]) Agustin Estrada-Pefia, ([double dagger]) Oscar Castro, * Perla A. Cabrera, * and Jose A. Oteo ([dagger])

* Universidad de la Republica, Montevideo, Uruguay; ([dagger]) Hospital de La Rioja, Logrono, Spain; and ([double dagger]) Universidad de Zaragoza, Zaragoza, Spain